Xenotransplantation may be clinically feasible once the mechanisms of rejection are understood and graft survival can be achieved without compromising the recipient to the extent that systemic toxicity is encountered. Thrombotic and inflammatory reactions to porcine bone marrow (BM)-derived cells and vasculature are linked to difficulties in establishing mixed discordant chimerism in primates and the development of thrombotic microangiopathy in vascularized grafts. These responses may be associated with humoral immunity to xenogeneic grafts and intrinsic molecular barriers between the discordant species. The development of the GalT-KO pig and removal of the dominant xenoantigen has been a major advance in this area. However, there are still problems in inducing tolerance by generating mixed chimerism, either by vascularized thymic tissues or the bone marrow BM-derived cell approach; limited xenograft survival times and graft injury are still a concern. The goals of this project are directed at delineating mechanisms of the thrombotic sequelae associated with the GalT-KO pig-to-baboon xenotransplant model. We will identify and characterize porcine antigenic targets of both natural and elicited xenoreactive antibodies directed against Gal negative xenografts in vivo by MALDI-TOF Mass Spectrometry. Vascular markers of thrombotic injury will be also determined by porcine gene mini-arrays. Protein expression profiling will be undertaken to validate these vascular markers of xenograft rejection. The role of antithrombotic interventions will be determined addressing both pharmacological and genetic modalities. Antithrombin agents will be administered to baboon recipients of porcine cell infusions and renal grafts, as dual anti-thrombotic and anti- inflammatory agents, at the time of graft implantation and with rejection episodes. We will also study treatment with solCD39 (an ectonucleotidase) and ATL146e (an adenosine receptor A2a agonist), in combination with this antithrombin strategy. Gene therapeutic vectors will be employed to over-express CD39, thrombomodulin and tissue factor pathway inhibitor in GalT-KO BM-derived cells, prior to their infusion into baboons. Outcomes with respect to inflammatory or thrombotic sequelae and engraftment will be examined. We will finally evaluate transgenic approaches to over-express CD39 and the natural human anticoagulant factors in mice and pigs. Our studies will be judged successful if novel and clinically relevant antithrombotic strategies can be then developed and applied.